Glutamine-free mammalian expression of recombinant glycoproteins with uniform isotope labeling: an application for NMR analysis of pharmaceutically relevant Fc glycoforms of human immunoglobulin G1
Glutamine-free mammalian expression of recombinant glycoproteins with uniform isotope labeling: an application for NMR analysis of pharmaceutically relevant Fc glycoforms of human immunoglobulin G1
Mammalian cells are widely used for producing recombinant glycoproteins of pharmaceutical interest. However, a major drawback of using mammalian cells is the high production costs associated with uniformly isotope-labeled glycoproteins due to the large quantity of labeled l-glutamine required for their growth. To address this problem, we developed a cost-saving method for uniform isotope labeling by cultivating the mammalian cells under glutamine-free conditions, which was achieved by co-expression of glutamine synthase. We demonstrate the utility of this approach using fucosylated and non-fucosylated Fc glycoforms of human immunoglobulin G1.
[NMR paper] Sparse isotope labeling for nuclear magnetic resonance (NMR) of glycoproteins using 13C-glucose.
Sparse isotope labeling for nuclear magnetic resonance (NMR) of glycoproteins using 13C-glucose.
http://www.bionmr.com//www.ncbi.nlm.nih.gov/corehtml/query/egifs/https:--academic.oup.com-images-oup_pubmed.png Related Articles Sparse isotope labeling for nuclear magnetic resonance (NMR) of glycoproteins using 13C-glucose.
Glycobiology. 2020 Sep 08;:
Authors: Rogals MJ, Yang JY, Williams RV, Moremen KW, Amster IJ, Prestegard JH
Abstract
Preparation of samples for nuclear magnetic resonance (NMR) characterization of larger proteins...
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09-13-2020 09:18 AM
Methyl-selective isotope labeling using α-ketoisovalerate for the yeast Pichia pastoris recombinant protein expression system
Methyl-selective isotope labeling using α-ketoisovalerate for the yeast Pichia pastoris recombinant protein expression system
Abstract
Methyl-detected NMR spectroscopy is a useful tool for investigating the structures and interactions of large macromolecules such as membrane proteins. The procedures for preparation of methyl-specific isotopically-labeled proteins were established for the Escherichia coli (E. coli) expression system, but typically it is not feasible to express eukaryotic proteins using E. coli. The Pichia pastoris (P. pastoris)...
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06-05-2018 01:31 PM
[NMR paper] Stable isotope labeling approaches for NMR characterization of glycoproteins using eukaryotic expression systems.
Stable isotope labeling approaches for NMR characterization of glycoproteins using eukaryotic expression systems.
Related Articles Stable isotope labeling approaches for NMR characterization of glycoproteins using eukaryotic expression systems.
J Biomol NMR. 2018 Feb 28;:
Authors: Yanaka S, Yagi H, Yogo R, Yagi-Utsumi M, Kato K
Abstract
Glycoproteins are characterized by the heterogeneous and dynamic nature of their glycan moieties, which hamper crystallographic analysis. NMR spectroscopy provides potential advantages in dealing...
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03-02-2018 03:20 PM
Stable isotope labeling approaches for NMR characterization of glycoproteins using eukaryotic expression systems
Stable isotope labeling approaches for NMR characterization of glycoproteins using eukaryotic expression systems
Abstract
Glycoproteins are characterized by the heterogeneous and dynamic nature of their glycan moieties, which hamper crystallographic analysis. NMR spectroscopy provides potential advantages in dealing with such complicated systems, given that the target molecules can be isotopically labeled. Methods of metabolic isotope labeling in recombinant glycoproteins have been developed recently using a variety of eukaryotic production vehicles,...
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02-28-2018 03:32 PM
Expression and purification of (15)N- and (13)C-isotope labeled 40-residue human Alzheimer's ?-amyloid peptide for NMR-based structural analysis.
Expression and purification of (15)N- and (13)C-isotope labeled 40-residue human Alzheimer's ?-amyloid peptide for NMR-based structural analysis.
Expression and purification of (15)N- and (13)C-isotope labeled 40-residue human Alzheimer's ?-amyloid peptide for NMR-based structural analysis.
Protein Expr Purif. 2011 May 27;
Authors: Long F, Cho W, Ishii Y
Amyloid fibrils of Alzheimer's ?-amyloid peptide (A?) are a primary component of amyloid plaques, a hallmark of Alzheimer's disease (AD). Enormous attention has been given to the structural...
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06-07-2011 11:05 AM
Stable-isotope-assisted NMR approaches to glycoproteins using immunoglobulin G as a m
Stable-isotope-assisted NMR approaches to glycoproteins using immunoglobulin G as a model system.
Related Articles Stable-isotope-assisted NMR approaches to glycoproteins using immunoglobulin G as a model system.
Prog Nucl Magn Reson Spectrosc. 2010 May;56(4):346-59
Authors: Kato K, Yamaguchi Y, Arata Y
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10-19-2010 04:51 PM
Stable-isotope-assisted NMR approaches to glycoproteins using immunoglobulin G as a m
Stable-isotope-assisted NMR approaches to glycoproteins using immunoglobulin G as a model system
Publication year: 2010
Source: Progress in Nuclear Magnetic Resonance Spectroscopy, In Press, Accepted Manuscript, Available online 19 March 2010</br>
Koichi, Kato , Yoshiki, Yamaguchi , Yoji, Arata</br>
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08-16-2010 03:50 AM
Enhanced production and isotope enrichment of recombinant glycoproteins produced in c
Abstract NMR studies of post-translationally modified proteins are complicated by the lack of an efficient method to produce isotope enriched recombinant proteins in cultured mammalian cells. We show that reducing the glucose concentration and substituting glutamate for glutamine in serum-free medium increased cell viability while simultaneously increasing recombinant protein yield and the enrichment of non-essential amino acids compared to culture in unmodified, serum-free medium. Adding dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, further improves cell viability, recombinant...
Glutamine-free mammalian expression of recombinant glycoproteins with uniform isotope labeling: an application for NMR analysis of pharmaceutically relevant Fc glycoforms of human immunoglobulin G1
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